Nucleic acids, i.e., DNA and RNA, are well known as the so-called "genetic blueprint" of all cell types. Be they prokaryotes or eukaryotes, cells require DNA and RNA for the production of various proteins needed for viability.
Much of the current work being performed in the biological sciences requires manipulation of pure nucleic acids. Transformation, transfection, Northern and Southern blots, polymerase chain reactions, and so forth, all require a ready source of purified nucleic acid material. In addition, purified nucleic acids are used in applications such as "DNA fingerprinting" in forensic sciences, so-called "RFLP" analysis, and other applications where purified DNA or RNA is necessary.
Nucleic acids do not exist in free form in cells; rather they interact with various molecules and organelles in vivo forming, e.g., nucleo-protein complexes. Biological materials present nucleic acid material that is encapsulated in protein coats or in association with membranes. See, in this regard Rodriquez and Tait, Recombinant DNA Techniques, An Introduction (Benjamin Cummings, 1983), pg. 37-38.
Standard methods for isolation of DNA call for lysis of the sample (tissue or cell), by various methods, in connection with various enzymes such as proteinase K. These procedures yield a mix of DNA and macromolecules which must be separated. One classic method for DNA purification utilizes an extraction reagent containing organic solvents, such as phenol and chloroform, optionally with isoamyl alcohol. See, in this regard Murmur, J. Mol. Biol. 3: 208-218 (1961); Gross-Bellard, et al., Eur. J. Biochem. 36: 32-38 (1973); Blin, et al., Nucl. Acid. Res. 3: 2303-2308 (1976). In these methods, the nucleic acids are separated into the aqueous phase of a two phase organic/non-organic (i.e., water) system. The aqueous layer having a lower density than the organic portion of the reagent, rises to the top of a mixture, from which it can be removed or extracted.
While this methodology is a standard one, it requires expensive reagents and equipment to perform, and a good deal of time. Additionally, phenol and chloroform are both noxious to use, cause burns on contact to skin and mucus membranes, require cumbersome safety apparatus, such as hoods to prevent inhalation and have been implicated as potential carcinogens. In addition, contamination of the aqueous, DNA containing layer by the organic solvent (phenol) is a ubiquitous problem. This contamination renders the DNA useless for further manipulations without additional purification procedures. Awareness of this problem leads to a need for the investigator to exercise extreme caution, especially with respect to the interface between the organic and non-organic layers. This can, and frequently does, lead to decreased yields of nucleic acids, and additional processing steps, such as "back-extraction" (i.e., the extraction of the remaining, mostly organic material, with additional aqueous solvents).
The patent literature describes the use of techniques such as those described supra in connection with other biochemical inventions. For example, U.S. Pat. No. 4,623,627 teaches obtaining double stranded DNA using the phenol/chloroform methodology described supra.
Another method used to separate DNA is cesium chloride gradient ultracentrifugation. See, Glisin, et al., Biochemistry 13: 2633-2637 (1974). In this methodology, a DNA solution is mixed with a cesium chloride solution or a mixture of cesium chloride and ethidium bromide. The mixture is then centrifuged, resulting in a gradient of increasing salt concentrations. DNA molecules band at positions within the gradient corresponding to their buoyant density.
This methodology, however, requires the use of an ultracentrifuge as well as expensive chemicals, and a good deal of time. While of interest, it is not the optimum approach, as is evidenced by the number of papers teaching variations on this general technique. See, e.g., Meese, et al., Gene Anal. Tech. 4: 45-49 (1986), where guanidine HC1 is used in combination with the cesium chloride gradient technique.
A second group of methodologies, which may be referred to collectively as "column purification", is of more pertinence to the subject invention. In column purification, the nucleic acid containing sample is applied to a solid phase matrix. For example, Shoyab, et al., Meth. Enzymol. 68: 199-206 (1979) describe purification of genomic DNA using hydroxyapatite column chromatography. U.S. Pat. No. 4,649,119 teaches that hydroxyapatite may be used as a support to recover plasmids from Corynebacterium bacteria.
An additional type of column purification is taught by Potter, et al., Cancer Lett. 26: 335-341 (1985). This method uses a matrix of diethylamino ethyl (DEAE) sepharose, referred to hereafter as a "DEAE matrix".
A DEAE matrix presents a positively charged ion (N.sup.+) covalently bound to the matrix, together with a mobile counterion (C1.sup.-). These mobile counterions are available for exchange, under suitable conditions, with other negatively charged ions, such as proteins and nucleic acid molecules. DEAE matrices are quite common in column separation, as is taught by Maniatis, et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories), pp. 130 and 164.
The first step in ion exchange column separation is application of the sample to the column. Standard methodologies instruct the investigator to apply the sample without disturbing the matrix bed. This is followed by a washing step, generally using a low salt buffer. Potter, et al., supra, call for washing the sample containing column with a buffer of 10 mM Tris, 1 mM EDTA, and 0.1 M NaC1. Variations may be seen in, e.g., European Patent Application 270 017, to Molecular Biosystems, Inc., teaching washing with a salt strength of from 0.2 to 0.5 M.
The step as described in these references removes impurities that are present in the applied sample. The salt concentration is kept low (i.e., a weak ionic strength buffer is used) because the ions in the salt solution will complete with the DNA bound to the column.
Following the wash step in which impurities are eluted, DNA is collected from the column via, e.g., elution. Here, a higher concentration of salt is required, because the very effect avoided in the washing step is now desired. Hence, in Potter, supra, elution is at 1.0 M NaCl, while the European Patent Application referred to supra elutes at anywhere from 0.5 M to 1.0 M, depending upon the nature of the nucleic acid and the column used. The amount of elution buffer used is desirably kept as low as possible. In practice, however, this aim is in conflict with the need to use a larger amount of buffer so as to elute all or as much of the nucleic acids as possible.
Other references teach different elution reagent strengths. U.S. Pat. No. 4,389,396, to d'Hinterland, e.g., teaches gradient elution using from 0.0 to 0.5 M NaCl. U.S. Pat. No. 4,649,119 to Sinskey teaches elution using 1.0 M potassium phosphate. Indeed, the strongest elution solution currently observed in the art is 1.0 M.
The ion exchange purification methodologies elaborated upon supra all adhere to the teaching in the art that instructs one to apply the same to the matrix surface without disrupting the matrix surface.
It has now been found, surprisingly, that upon creating a uniformly mixed slurry upon application of the sample, the nucleic acids are uniformly mixed throughout the matrix, leading to effective interaction between the nucleic acid molecules and the ion exchange matrix. This, in turn leads to effective recovery of purified, high molecular weight nucleic acids.
It has also been found that by including an optional "priming step" in which a high strength ionic salt solution is applied to the ion exchange matrix before elution of the nucleic acid allows the nucleic acid to be recovered in a smaller volume of elution buffer. This, too, is a result not to be expected from the prior art.